Part:BBa_K4767011

Pars-RBS-arsR-RBS-luxR(△2-162)-TT-PluxⅠ-RBS-luxR(△2-162)-RBS-cymA-TT

Uses a factor Pars(BBa_K4767001) ,strong RBS(BBa_J34801), DNA binding transcriptional repressor arsR(BBa_J15101), luxR(△2-162)(BBa_K4767000) TT(BBa_B015) ,PluxⅠ(BBa_R0062) and cymA(BBa_K2706008).This composite part is the arsenic responding biosensor with the LuxR-based self amplifier.

Usage and Biology

In order to construct a positive feedback circuit which does not require 3OC6HSL produced by LuxI, we engineered LuxR by deleting 2-262 amino acids in the N-terminal domain (AHL binding domain) and reserving a C-terminal domain with the function of activating transcription, obtaining a resulting regulator LuxR(△2-162). LuxR(△2-162) can active the gene transcription driven by the lux promoter in the absence of AHL. To construct the amplifier, we cloned gfp and LuxR(Δ2-162) behind the lux promoter. In this design, LuxR(Δ2-162) functions in a positive feedback loop as it can bind to the PluxI promoter and activate its own transcription.

File:BBa K4767011.jpg

After the modification of the LuxR(Δ2-162), we applied the amplifier into the arsenic-response system we build. The transcriptional activator, LuxR(Δ2-162), was used to replace cymA as reporter, and it was regulated by the arsR-Pars circuit, while cymA together with a second LuxR(Δ2-162) was placed under the promoter PluxI, which was activated by LuxR(Δ2-162). When arsenic is present, it activates the expression of LuxR(Δ2-162) in the first circuit, which turns on the expression of cymA and LuxR(Δ2-162) from the following circuit. The second LuxR(Δ2-162) activates its own expression as well as that of cymA and forms a positive feedback loop to enhance the output signal from cymA. These two parts work together as the arsenic EAS-based sensor with the positive feedback amplifier. Our results suggested that, compared with the sensor without positive feedback, the one with a positive feedback amplifier functions well in enhancing the iron reduction rates, increasing the detection range, and improving sensitivity. By introducing the positive feedback amplifier into the arsenic sensor, the output signal was enhanced so much that the specificity of the sensor toward arsenic was also significantly increased.

File:BBa K47670111.jpg Fig. Iron reduction rates in different arsenic levels. Plus sign refers to the strains with the amplifier.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 270
  Illegal BglII site found at 1616
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 929
  

References

Nistala, G.J., et al., A modular positive feedback-based gene amplifier. J Biol Eng, 2010. 4: p. 4.

Xiaoqiang Jia, Bu Rongrong, Zhao Tingting, et al. Sensitive and Specific Whole-Cell Biosensor for Arsenic Detection[J]. Applied and environmental microbiology, 2019, 85(11): 1.